The present invention relates to a mutant RNA polymerase obtained from a bacteriophage that has improved thermal stability and/or specific activity in comparison with a wild type under high-temperature conditions, and more particularly, to T7 RNA polymerase. Although examples of bacteriophages that can infect Escherichia coli include T3, T7, φI, φII, W31, H, Y, A1, croC21, C22 and C23, RNA polymerase encoded by a T7 phage is T7 RNA polymerase.
The first characteristic of T7 RNA polymerase is high selectivity with respect to a promoter sequence. Although T7 RNA polymerase binds to its own unique promoter sequence, it does not bind with other promoter sequences even if they are promoter sequences of other bacteriophages. Due to this high selectivity, RNA polymerase transcription reactions can be reliably improved with respect to its own genome instead of the host genome.
Next, different from other polymerases, T7 RNA polymerase has a series of functions that enable it to recognize a promoter, begin transcription, elongate the RNA transcription product and terminate transcription without requiring a cofactor, and is able to elongate RNA five times faster than E. coli RNA polymerase.
Moreover, since it is a single-stranded protein having a molecular weight of 98.6 kDa and 883 amino acids, it enables inexpensive, large-volume production of enzymes.
As a result of having the advantages described above, T7 RNA polymerase is aggressively used in various fields, examples of which include in vitro transcription and a high expression system in E. coli (U.S. Pat. No. 4,952,496: Patent Document 1), a cell-free protein synthesis system, a base sequencing method (Japanese Unexamined Patent Publication No. H11-18799: Patent Document 2), and an isothermal nucleic acid amplification method. The following provides a detailed explanation of the TRC method, which is a type of isothermal nucleic acid amplification method (Japanese Unexamined Patent Publication No. 2000-14400: Patent Document 3, and Ishiguro, T. et al., Analytical Biochemistry, 314, 77-86 (2003): Non-Patent Document 1).
The TRC method is a method for amplifying a target RNA containing a specific RNA sequence by utilizing a concerted action between DNA-dependent RNA polymerase and reverse transcriptase. Namely, by use of a primer specific to the target RNA which comprises a T7 promoter sequence, reverse transcriptase and ribonuclease H, a double-stranded DNA comprising the promotor sequence is synthesized, and then RNA composed of the specific RNA sequence is synthesized by use of a T7 RNA polymerase. The synthesized RNA is used as a template for synthesizing double-stranded DNA that contains the aforementioned promoter sequence, thereby the aforementioned reaction is carried out in the manner of chain reaction. Differing from the case of amplifying by the PCR method, since amplification of nucleic acids by the TRC method enables the reaction to be carried out at a constant temperature, it has the advantage of eliminating the need for complex temperature control. However, when nucleic acid amplification is carried out with the TRC method using wild-type T7 RNA polymerase, reduction in nucleic acid amplification efficiency is observed at temperatures of 46° C. or higher due to a decrease in activity of the T7 RNA polymerase. Consequently, nucleic acid amplification using the current TRC method is typically carried out under comparatively low temperature conditions in the order of 40 to 45° C. However, RNA has a tendency of assuming a complex, higher order structure under low temperature conditions, and this has made it difficult to design primers capable of highly sensitive detection in the TRC method. Consequently, there has been a need for T7 RNA polymerase that demonstrates high thermal stability and/or high specific activity even under temperature conditions of 46° C. or higher.
Since systems have been established for measuring T7 RNA polymerase activity (Ikeda, R. A. et al., Biochemistry, 31, 9073-9080 (1992): Non-Patent Document 2, and Ikeda, R. A. et al., Nucl. Acid Res., 20, 2517-2524 (1992): Non-Patent Document 3), several RNA polymerases have been produced that have various improved functions by mutation. Examples of such RNA polymerases include an enzyme the promoter sequence which it recognizes had been altered through amino acid substitution (U.S. Pat. No. 5,385,834: Patent Document 4), an enzyme having enhanced specific activity and thermal stability at high temperatures (Japanese Unexamined Patent Application Publication (Translation of PCT Application) No. 2003-525627: Patent Document 5), and an enzyme having enhanced 3′-deoxyribonucleotide uptake function through amino acid deletion and substitution (Japanese Unexamined Patent Publication No. 2003-61683: Patent Document 6).